Amplification refractory mutation system PCR assays for the detection of variola and Orthopoxvirus

Abstract

PCR assays that can identify the presence of variola virus (VARV) sequences in an unknown DNA sample were developed using principles established for the amplification refractory mutation system (ARMS). The assay's specificity utilised unique single nucleotide polymorphisms (SNP) identified among Orthopoxvirus (OPV) orthologs of the vaccinia virus Copenhagen strain A13L and A36R genes. When a variola virus specific primer was used with a consensus primer in an ARMS assay with different Orthopoxvirus genomes, a PCR product was only amplified from variola virus DNA. Incorporating a second consensus primer into the assay produced a multiplex PCR that provided Orthopoxvirus generic and variola-specific products with variola virus DNA. We tested two single nucleotide polymorphisms with a panel of 43 variola virus strains, collected over 40 years from countries across the world, and have shown that they provide reliable markers for variola virus identification. The variola virus specific primers did not produce amplicons with either assay format when tested with 50 other Orthopoxvirus DNA samples. Our analysis shows that these two polymorphisms were conserved in variola virus genomes and provide a reliable signature of Orthopoxvirus species identification.

Fig. 1

Diagram (not to scale) illustrating PCR primers, Orthopoxvirus genes and their predicted PCR products. Open reading frames are shown as open arrows, correctly orientated in relation to the vaccinia virus Copenhagen genome and incomplete adjacent ORFs are shown with slashed lines. PCR primers are shown as black filled arrows (generic pair) or dashed arrows (variola virus specific). The predicted amplicon size for each primer pair is shown with dotted lines underneath each ORF.

Fig. 2

The specificity of multiplex PCR assays with a panel of DNA from different Orthopoxvirus species. Multiplex assays were performed on DNA prepared from purified viruses as described in Section 2. DNA samples included; (1) vaccinia virus Copenhagen, (2) vaccinia virus MVA, (3) camelpox virus CP1, (4) cowpox virus Brighton, (5) cowpox virus EP1, (6) cowpox virus Norway, (7) ectromelia virus MP1, (8) monkeypox virus Z1, (9) racoonpox virus VR838, (10) variola virus synthetic control template, (11) water and (M) 100 bp ladder (Roche). Ten microliters of PCR product was loaded on a 1% agarose gel.

Fig. 3

Testing the variola virus (VARV) primer specificity using an ARMS PCR. Reaction mixes included either variola virus specific primer A13L-3 or A36R-3, matched with a single Orthopoxvirus generic forward primer (i.e., A13L-1 or A36R-1, respectively). One nanogram of viral DNA from (1) variola virus Congo, (2) camelpox virus Somalia, (3) cowpox virus Brighton, (4) monkeypox virus 79-I-005, (5) vaccinia virus Copenhagen or (6) mock infected BSC40 cell DNA was added to 25 μl of PCR mix. DNA markers (100 bp ladder, Roche) are shown in the last lane on each gel (M). PCR primers were used in equal concentrations. Primer-dimers are present at the base of the A36R1 and three gel.

Fig. 4

Multiplex PCR performed on panels of variola virus genomic DNA samples extracted from virus infected BSC40 cells. DNA samples used for A13L multiplex a and the A36R multiplex included variola strains; (1) Congo, (2) Heidelberg, (3) Eth72-16, (4) v70-228, (5) v68-59, (6) Minnisota 124, (7) Juba, (8) Nepal 73, (9) K1629, (10) Harper, (11) Butler, (12) Horn, (13) Hinden, (14) 7125, (15) Kembula, (16) SAF65-103, (17) Higgins, (18) Iran 2602, (19) v77-1252, (20) Solaiman, and control strains included (21) camelpox virus (CMLV) Somalia, (22) cowpox virus (CPXV) Brighton, (23) monkeypox virus (MPXV) 79-I-005, (24) vaccinia virus (VACV) Copenhagen and (25) BSC40 mock infected cells. DNA samples used with the A13L multiplex b PCR included variola strains; (1) Garcia, (2) Yamada, (3) Lahore, (4) Lee, (5) V70-222, (6) Shah, (7) Kali Mathu, (8) Rumbec, (9) v77-1605, (10) 102, (11) v72-119, (12) v68-258, (13) 7124, (14) v73-225, (15) Ethiopia 17, (16) New Delhi, (17) Stillwell, (18) Harvey, (19) Nur Islam, (20) v66-39, (21) 72-143, (22) Variolator 4, (23) Somalia strains and control DNA from (24) vaccinia virus Copenhagen. DNA markers (100 bp ladder, Roche) are shown in lanes marked (M). Ten microliters of each PCR was run on a 1.5% agarose gel and stained with ethidium bromide to visualize DNA bands by UV illumination.

Fig. 5

Performance of a variola virus specific multiplex PCR assays with DNA extracted from clinical samples including variola viruses; (1) Solaiman (1.82 ng), (2) Kudano (1.23 ng), (3) Herrlich (8.59 ng), (4) Hembula (0.60 ng), (5) Variolator 4 (0.11 ng), (6) Parvin (3.83 ng), (7) Mannan (11.03 ng), (8) Varicella zoster V01-I-01 (2.37 ng) and (9) variola virus Congo infected BSC40 cell extract (1.85 ng) in a 25 μl multiplex PCR assay containing A13L1, two three three primers per tube. Amplified products were visualized by agarose gel electrophoresis and are indicated with arrows. A 100 bp ladder (Roche) is shown marked (M).